A major challenge for treating cocaine addiction is the propensity for abstinent users to relapse. Two important triggers for relapse are cues associated with prior drug use and stressful life events. During my postdoctoral training, I studied mechanisms in the nucleus accumbens (NAc) underlying the withdrawal-dependent intensification (incubation) of cue-induced cocaine craving. As a PI, I will incorporate the effect of chronic stress into my studies. To date, the majority of studies investigating stress-induced relapse vulnerability have examined the effects of acute stressors on the reinstatement of previously extinguished drug seeking behavior, a model which may not accurately depict the situation of addicts, who typically do not undergo extinction training and who may relapse after a long drug-free period. Thus, there is an urgent need for animal models that explore interactions between the effects of chronic stress and drug withdrawal on cue-induced cocaine craving. To address this need, I will study the effect of chronic stress during abstinence on relapse vulnerability using the 'incubation of cocaine craving' model mentioned above, in which cue-induced drug seeking in rats progressively intensifies during the first months of withdrawal from extended-access drug self-administration. By combining my long-standing interest in addiction with my emerging interest in the neurobiology of stress, I have developed a research program that will be independent of my Mentor's research and will allow me to pursue three long-term goals: developing animal models with better face validity for addiction, using in vivo models which preserve neuronal circuits and enable correlation of cellular and behavioral results, and ultimately contributing to development of pharmacotherapies to help recovering addicts maintain abstinence. This Research Plan, along with the Training Plan I have developed with my Mentor (Dr. Marina Wolf) and co- Mentors (Dr. J. Amiel Rosenkranz and Dr. Janice Urban), will enable me to achieve my career goal of becoming an independent researcher in an academic setting. My project draws on previous work from my co-Mentors on the basolateral amygdala (BLA), a critical region for behavioral responses to stress. Dr. Rosenkranz's lab has shown that chronic stress enhances excitatory drive in the BLA, while Dr. Urban's lab has found that repeated activation of NPY receptors in the BLA produces long-lasting stress resilience. Furthermore, recent work from the Dong lab (2013) has shown that glutamate projections from the BLA to the NAc are critical for the expression of incubated cue-induced cocaine seeking. Combining these lines of work, my central hypothesis is that cocaine withdrawal and chronic stress exposure produce a synergistic increase in BLA neuronal activity which facilitates incubation of craving, whereas treatments promoting stress resilience will prevent this facilitation by producing opposing neuroadaptations in the BLA. My preliminary data show that chronic food restriction stress during withdrawal does in fact accelerate the incubation of cocaine craving. In Aim 1 (K99), I will determine the persistence of this effect and whether it generalizes to repeated restraint stress, the form of stress used in many prior studies of the BLA. In Aim 2 (K99/R00), I will test my hypothesis that cocaine withdrawal and stress exert synergistic effects in the BLA by comparing intrinsic excitability, synaptic transmission, and morphological features of BLA neurons of rats that self-administer saline or cocaine and then either undergo repeated stress or control conditions during withdrawal. In Aim 3 (K99/R00), I will determine if NPY receptor activation in the BLA opposes the chronic stress-induced facilitation of incubation of cocaine seeking and employ biochemical methods to identify mechanisms in the BLA through which NPY signaling mediates these protective effects. During my prior training, I secured individual pre- and postdoctoral NRSA funding, published in high impact journals, and gained expertise in behavioral and biochemical approaches. However, to undertake my proposed experiments, I need training in electrophysiology and stress models, as well as more experience with non-bench skills required for independence. To accomplish these goals, I have put together an exceptional team of advisors. My Primary Mentor Dr. Wolf is an expert in cocaine-induced neuronal plasticity who will oversee my training. Dr. Rosenkranz (co-Mentor) uses electrophysiological approaches to study the effects of stress on affective behavior and physiology of the amygdala. He will train me to conduct in vivo intracellular recordings in BLA neurons and then assess their morphology. Dr. Urban (co-Mentor) is a neuroendocrinologist who studies adaptations in the amygdala contributing to stress resilience. She will provide training on models of stress resilience and NPY signaling. I will also be guided by a Research Committee composed of my Mentor, co-Mentors, and two other electrophysiologists at my University, Drs. Kuei-Yuan Tseng and Anthony West. All of these individuals have a strong history of collaborative interactions. Together, we have developed a Training Plan that utilizes regular individual meetings with Mentors, bi-annual meetings of the Research Committee, coursework (including seminars and journal clubs), and an institutional postdoctoral development program to develop the non-bench skills needed for my transition from a postdoctoral fellow to an independent investigator in an academic setting. This training plan is strengthened by an outstanding institutional environment offering excellent facilities and a concentration of addiction researchers in a collaborative and supportive setting.